All about bacteria, bacteria culture

1. BACTERIA
(Namra Ibrar)
Lecturer
RLCP

2. General and cellular morphology
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Bacteria are unicellular prokaryotic microorganism. The cell structure is
simpler than that of other organisms as there is no nucleus or membrane
bound organelles. Instead their control center containing the genetic
information is contained in a single loop of DNA.
Bacteria represent a large and diverse group of microorganisms that can
exist as single cells or as cell clusters. Moreover, they are generally able to
carry out their life processes of growth, energy generation and reproduction
independently of other cells. In these respects they are very different from
the cells of animals and plants, which are unable to live alone in nature and
can exist only as part of a multicellular organism.
They are capable of growing in a range of different environments and can
not only cause contamination and spoilage of many pharmaceutical
products but also a range of different diseases.

4. Identification parameters for bacteria
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Primary identification parameter for bacteria are
Shape
Size
Aggregation properties
Staining properties
Imaging methods

5. Cell shape
1. Cocci
2. Bacilli
3. Vibrios
4. Spirilla
5. Spirochetes
6. Actinomycetes
7. Mycoplasmas.

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Cocci (from kokkos meaning berry) are small, spherical or oval bacteria having one
of several distinct arrangements based on their planes of division.
Division in one plane produces either a diplococcus or streptococcus arrangement.
diplococcus: cocci arranged in pairs
streptococcus: cocci arranged in chains
Division in two planes produces a tetrad arrangement.
tetrad: cocci arranged in squares of 4

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Division in three planes produces a sarcina arrangement.
sarcina: cocci in arranged cubes of 8
Division in random planes produces a staphylococcus arrangement.
staphylococcus: cocci arranged in irregular, often grape-like clusters

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2. Bacilli (from baculus meaning rod) are rod shaped bacteria. Bacilli all divide in
one plane producing a bacillus, streptobacillus, diplobacillus or coccobacillus
arrangement.
Bacillus: single bacilli
streptobacillus: bacilli arranged in chains
coccobacillus: oval and similar to a coccus
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10. 3. Vibrios are comma shaped curved rods and derive their name from their
characteristics vibratory motility.

11. 4. Spirilla are rigid spiral forms.
5. Spirochetes (from speira meaning coil and chaite meaning hair) are flexuous
spiral forms

12. 6. Actinomycetes are rigid branching filamentous bacteria, so called because of a
fancied resemblance to the radiating rays of the sun when seen in tissue lesions (from
actis meaning ray and mykes meaning fungus).
7. Mycoplasmas are bacteria that are cell wall deficient and hence do not possess a
stable morphology. They occur as round or oval bodies and as interlacing filaments.

13. Size of bacteria
The average diameter of spherical bacteria is 0.5-2.0 µm. For rod-
shaped or filamentous bacteria, length is 1-10 µm and diameter is
0.25-1 .0 µm.
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Bacteria are the smallest free - living organisms, their size being measured in
micrometres.The limit of resolution with the unaided eye is about 200 microns.
Bacteria vary in size from a cell as small as 0.1 – 0.2 μm in diameter to those that
are > 5 μm in diameter.
Bacteria this large, such as Thiomargarita namibiensis, are extremely rare.
The majority of bacteria are 1– 5 μ m long and 1 – 2 μ m in diameter.

14. Imaging method
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The morphological study of bacteria requires the use of microscopes.
The types of microscope are
Light or optical microscope
Phase contrast microscope
Dark field/ Dark ground microscope
Electron microscope

15. Light or optical microscope
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They are of two types namely Simple and Compound Microscope
Simple Microscope consists of a single lens. A hand lens is an example of a simple
Microscope.
Compound Microscope consists of two or more lenses in series. The image formed
by the first lens is further magnified by another lens.
Bacteria may be examined under the compound microscope, either in the living
state or after fixation and staining.
Examination of wet films or hanging drops indicates the shape, arrangements,
motility and approximately size of the cells.
But due to lack of contrast details cannot be appreciated.

16. Phase contrast microscope
This imposes the contrast and makes evident the structure within the cells that differ
in thickness or refractive index.
The difference in the refractive index between bacteria cells and the surrounding
medium makes them clearly visible.
Retardation, by a fraction of a wavelength, of the rays of light that pass through the
object, compared to the rays passing through the surrounding medium, produces
phase difference between the two types of rays.

17. Dark field / Dark ground microscope
Another method of improving the contrast is the dark field microscope in
which reflected light is used instead of the transmitted light used in the
ordinal microscope.
The contrast gives an illusion of increased resolution, so that very slender
organisms such as spirochete, not visible under ordinary illumination, can be
clearly seen under the dark field microscope.

18. Electron Microscope
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Beams of electron are used instead of beam of light, used in light
microscope.
The object which is held in the path of beam scatters the electrons and
produces an image which is focused on a fluorescent viewing screen.
Gas molecules scatter electron, therefore it is necessary to examine the
object in a vacuum

19. Aggregation properties
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Many species exhibit distinctive aggregation states
E.g long chains, irregular clusters or regular clusters.
These aggregation states are determined by the orientation of the cell
division plan to the axis of the cell.
These aggregates are relatively stable and characteristic of species, can
be disrupted mechanically without loss of cell viability.

20. Staining properties
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Live bacteria do not show the structural detail under the light microscope
due to lack of contrast.
Staining techniques are used to produce colour contrast.
Routine methods of staining of bacteria involve dying and fixing smears
procedures that kill them.

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Bacteria have an affinity to basic dyes due to acidic nature of their
protoplasm. The commonly used staining techniques are
Simple stain
Negative stain
Impregnation stain
Differential stain
Gram positive staining
Gram negative staining

22. Cellular morphology of
bacteria

23. Structure of bacteria

24. Cellular morphology
Cell wall
The bacterial cell wall is an extremely important structure, being essential
for the maintenance of the shape and integrity of the bacterial cell.
The primary function of the cell wall is to provide a strong, rigid structural
component that can withstand the osmotic pressures caused by high
chemical concentrations of inorganic ions in the cell.
Most bacterial cell walls have in common a unique structural component
called peptidoglycan (also called murein or glycopeptide); exceptions
include the mycoplasmas, extreme halophiles and the archaea.
Peptidoglycan is a large macromolecule containing glycan (polysaccharide)
chains that are cross- linked by short peptide bridges.

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Bacteria can be divided into two large groups, Gram- positive and Gram- negative, on
the basis of a differential staining technique called the Gram stain.
Essentially, the Gram stain consists of treating a film of bacteria dried on a
microscope slide with a solution of crystal violet, followed by a solution of iodine;
these are then washed with an alcohol solution.
In Gram - negative organisms the cells lose the crystal violet– iodine complex and are
rendered colourless, whereas Gram- positive cells retain the dye.
Regardless, both cell types are counter - stained with a different coloured dye, e.g.
carbolfuchsin, which is red

26. Cell membrane
Biochemically, the cytoplasmic membrane is a fragile, phospholipid bilayer with
proteins distributed randomly throughout.
These are involved in the various transport and enzyme functions associated with
the membrane.
A major difference in chemical composition between prokaryotic and eukaryotic
cells is that eukaryotes have sterols in their membranes (e.g. cholesterol) whereas
prokaryotes do not.
The cytoplasmic membrane serves many functions, including transport of nutrients,
energy generation and electron transport; it is the location for regulatory proteins
and biosynthetic proteins, and it acts as a semipermeable selectivity barrier
between the cytoplasm and the cell environment.
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27. Importance of Cell Membrane
1. It mostly acts as an‘osmotic barrier’ , and usually contains permeases that are
solely responsible for the viable transport of nutrients and chemicals both in and
outside the cell .
2. It essentially contains the enzymes that are intimately involved in the biosynthesis
of membrane lipids together with a host of other macromolecules belonging to the
bacterial cell wall .
3) It pre-dominently comprises of the various components of the energy generation
system.
There is evidence to demonstrate and prove that the cell membrane has particular
‘attachment sites’ exclusively meant for the replication and segregation of the
bacterial DNA and the plasmids.

28. Cytoplasm
The cytoplasm is a Colloidal system containing a variety of organic and inorganic
solutes containing 80% Water and 20% Salts, Proteins.
Based upon various intensive and extensive investigations carried out on the bacterial
cell, one may observe that the major cytoplasmic contents of it essentially include not
only the nucleus but also ribosomes, proteins, plasmids, water-soluble components, and
reserve material.
It has also been observed that a plethora of bacteria do contain extrachromosomal DNA
i.e., DNA that are not connected to the chromosomes.

29. Ribosomes
Thespecific cytoplasmic area which is strategically located in the cell material
bound by the cytoplasmic membrane having granular appearance and invariably rich
in the macromolecular RNA-protein bodies is termed as ribosome.
Ribosomes may exist singly, in clusters called polyribosomes, or on the surface of
rough endoplasmic reticulum.
In protein synthesis, they are the most favoured site of messenger RNA attachment
and amino acid assembly in the sequence ordered b the genetic code carried by
mRNA.

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Interestingly, there are certain ribosomes that are found to be virtually‘free’ in the
cytoplasm.
There are some, particularly those critically involved in the synthesis of proteins
require to be transported out of the cell, get closely linked to the inner surface of the
cytoplasmic membrane.
The number of ‘ribosomes’ varies as per the ensuing‘rate of protein synthesis’, and
may reach even up to 15,000 per cell.
In fact, greater the rate of proteins synthesis, the greater is the rate of prevailing
ribosomes
In a situation when these ‘ribosomes’ are specifically associated with the mRNA in
the course of active protein synthesis, the resulting product is termed as ‘polysomes’.
‘antibiotics’like chloramphenicol, erythromycin, gentamycin, and streptomycin,

31. The nucleoid
Electron micrographs of the bacterial nucleus under investigation evidently depict it as a
region very tightly and intimately packed with fibrillar DNA
i.e., consisting of very small
filamentous structure.
Investigations with respect to a large cross-section of bacterial species revealed that the
‘bacterial nucleus’ essentially contains a distinct singular molecule of definite circular
shape, and having a double-stranded DNA.

32. Plasmid
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The plasmids are autonomous DNA molecules of varying size localized in the cytoplasm.
Large plasmids are usually present in one to two copies per cell, whereas small ones
may be present in 10, 40, or 100 copies.
Plasmids are not essential to a cell’s survival.
Many of them carry genes that code for certain phenotypic characteristics of the host cell.
The following plasmid types are medically relevant:
Virulence plasmids.
Carry determinants of bacterial virulence, e.g., enterotoxin genes or hemolysin
genes.
2. Resistance plasmids
Carry genetic information bearing on resistance to anti-infective agents

33. Cell surface components
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The surface of the bacterial cell is the portion of the organism that interacts
with the external environment most directly.
As a consequence, many bacteria deploy components on their surfaces in a
variety of ways that allow them to withstand and survive fluctuations in the
growth environment

34. Flagella
Bacterial motility is commonly provided by flagella, long ( c. 12 μm ) helical- shaped
structures that project from the surface of the cell.
The filament of the flagellum is built up from multiple copies of the protein flagellin.
Where the filament enters the surface of the bacterium, there is a hook in the
flagellum, which is attached to the cell surface by a series of complex proteins called
the flagellar motor.
This rotates the flagellum, causing the bacterium to move through the environment.
The numbers and distribution of flagella vary with bacterial species.
Some have a single, polar flagellum, whereas others are flagellate over their entire
surface (peritrichous); intermediate forms also exist.

36. Fimbriae and Pili
Fimbriae are structurally similar to flagella, but are not involved in motility.
Although they are straighter, more numerous and considerably thinner and shorter (3 μ m)
than flagella, they do consist of protein and project from the cell surface.
There is strong evidence to suggest that fimbriae act primarily as adhesins, allowing
organisms to attach to surfaces, including animal tissues in the case of some
pathogenic bacteria, and to initiate biofilm formation.
Fimbriae are also responsible for haemagglutination and cell clumping in bacteria.
Among the best characterized fimbriae are the type I fimbriae of enteric (intestinal)
bacteria.

37. Pili
Pili are morphologically and chemically similar to fimbriae, but they are
present in much smaller numbers ( < 10) and are usually longer. They are
involved in the genetic exchange process of conjugation .

38. CLASSIFICATION OF BACTERIA

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There are many methods to classify micro-organisms, which include;
Binomial classification
Classification according to Temperature
Classification according to Gram staining
Classification according to Presence of Oxygen
Classification according to Morphology of bacteria
Classification of Microorganisms (Taxonomy)

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Binomial classification
KINGDOM the highest level in classification
PHYLUM
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CLASS
ORDER
FAMILY
GENUS
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SPECIES organisms sharing a set of biological traits and reproducing only with
their exact kind
Further classifications especially with bacteria:
Strain—organisms within a species varying in a given quality
Type—organisms within a species varying immunologically

41. Kingdom: Eubacteria
Phylum: Proteobacteria
Class: Gammaproteobacteria
Order: Enterobacteriales
Family: Enterobacteriaceae
Genus: Escherichia
Species: E. coli
For example;
Escherichia coli

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Classification according to Temperature
Psychrophiles (Philic) ; Can survive under 15-25 C° . e.g. Bacillus psychrophilus
Mesophiles ; Can survive under 25-45 C° .e.g.; E.coli
Thermophiles ; Can survive under 45-60 C° . e.g. ; Bacillus stearothermophilus
Classification according to Gram Staining
Gram Positive Bacteria, e.g.; Staphylococcus aureus
Gram Negative Bacteria, e.g. ; E. coli

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Classification according to Presence of Oxygen
Obligate aerobic; Which Oxygen is the primary needs. e.g.; Mycobacterium
tuberculosis
Factitive anaerobic; Can survive with or without oxygen. e.g.; E.coli
Anaerobic; Which can not survive in the presence of Oxygen. e.g.: Clostridium tetani
Microaerophilic; Which need less amount of Oxygen. e.g. ; Neisseria gonorrhea
Aerotolerant; Do not required oxygen , nether dies in the presence of oxygen. e.g. ;
Lactobacillus

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Classification according to Morphology
Cocci ; Round shape , Diplo cocci ( two cocci), staphylococci (cluster of cocci),
Streptococci (chain of cocci)
Bacilli ; Rod shape
Vibrios ; Comma shape
Spirilla ; Flexible spiral shape
Spirochetes ; Spring type shape
Actinomycetes ; Branching filamentous bacteria
Mycoplasma ; Cell wall less bacteria

45. BACTERIAL GROWTH

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Bacteria‘s growth can be take place by binary fission and during that so
many phases happen and different events takes place.
Three type of growth curves:
Growth curve
Synchronous growth
Bacterial growth in vivo

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Bacterial growth is regulated by nutritional environment. When suitable environment is
there that time bacterium is incubated,its growth leads to increase in number of cells
which allow definite course.
The growth curve has got four phases:
Lag phase
Log phase(logarithmic) or exponential phase
Stationary phase
Decline phase
GROWTH CURVE

49. 1. LAG PHASE ( 0-4 HOURS)
During lag phase, bacteria adapt themselves to growth conditions.
It is the period where the individual bacteria are maturing and not yet able to divide.
During the lag phase of the bacterial growth cycle, synthesis of RNA, enzymes and other
molecules occurs.
During the lag phase cells change very little because the cells do not immediately
reproduce in a new medium.
This period of little to no cell division is called the lag phase and can last for 1 hour to
several days.
During this phase cells are not dormant.

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(sometimes called the log phase or the logarithmic phase)
it is a period characterized by cell doubling.
The number of new bacteria appearing per unit time is proportional to the present
population.
If growth is not limited, doubling will continue at a constant rate so both the number
of cells and the rate of population increase doubles with each consecutive time
period.
For this type of exponential growth, plotting the natural logarithm of cell number
against time produces a straight line. The slope of this line is the specific growth rate
of the organism, which is a measure of the number of divisions per cell per unit time.
The actual rate of this depends upon the growth conditions, which affect the
frequency of cell division events and the probability of both daughter cells surviving.
Under controlled conditions, cyanobacteria can double their populating four times a
2.LOG PHASE OR EXPONENTIAL (EV : 8HR)

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The "stationary phase" is due to a growth-limiting factor; this is mostly depletion of a
nutrient, and/or the formation of inhibitory products such as organic acids.
An awkward but unfortunately widespread explanation is that the stationary phase
results from a situation in which growth rate and death rate have the same values
newly formed cells per time = dying cells per time
but this is not logical, and it is better to forget this.
Such an explanation would not be in accordance with the observed substrate
depletion and also could never explain the rather “smooth,” horizontal linear part of
the curve during the stationary phase. Death of cells as a function of time is rather
unpredictable and very difficult to explain.
Another not really logical explanation of the stationary phase is that there isn’t
anymore enough space for the cells.
3.STATIONARY PHASE (FEW HOURS TO
DAYS)

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Another not really logical explanation of the stationary phase is that there isn’t
anymore enough space for the cells.
However, under the microscope you will see that there is still plenty of water
between the cells.
Only in an agar colony with densely packed cells space is obviously limiting.

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Bacteria run out of nutrients and die although number of cells remain constant.
The decline phase is brought by exhaustion of nutrients, accumalation of toxic
products and autolytic enzymes.
Sometimes a small numbers of survivors may persist for month even after death of
majority of cells these few surviving cells probably grow at expence of nutrients
released.
4. DECLINE PHASE (FEW HOURS - DAYS)

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Bacteria grow nonsynchronously in ordinary culture medium, i.e at any moment
cells are present in different stage of growth cycle.
When all bacterial cells in culture medium divide simultaneously growth thus
obtained is known as synchronous growth.
Such growth is required for studing the sequence of event occuring in single cell
like studies on DNA synthesis or susceptibility of cell to lethal agent.
SYNCHRONOUS GROWTH

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There exists a significant difference of bacteria growth in human body and artificial
culture medium.
They grow much faster in vitro than in vivo.
Various factor in vivo include:
nutritional status of body
generation time
defense mechanism
redox potential
hydrogen iron concentration
localization of nutrients
BACTERIAL GROWTHIN-VIVO

56. GROWTH
FACTORS

57. Factors affecting Microbial growth
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1.Physical factors
pH
Temperature
Osmotic pressure
Hydrostatic pressure
Moisture
Radiation
Oxygen concentration

58. 2.Nutritional factors
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Carbon
Nitrogen
Sulfur
Phosphorus
Trace elements
vitamins

59. 1.pH
The microorganisms are divided into different categories with respect to their pH
range.
Acidophiles (0.1 – 5.4) e.gLactobacillus
Neutrophiles (5.4 – 8.0) e.gBacterial pneumonia
Alkaliphiles (7.0 – 11.5) e.gAgrobacterium
Physical factors

60. 2. Temperature
Psychrophiles “cold loving bacteria” (-20C° TO 15 C°)
Obligate psychrophiles
These can not grow above 20C° e.gBacillus globisporus, Oscillatoria, Chlamydomonas
nivalis, Methanogenium,
Facultative psychrophiles
It grows best below 20 C° but can also grow above e.gXanthomonas pharmicola
Mesophiles 25 – 40C°
e.g all human pathogens
Thermophiles “Heat loving” (50 -60C°) Obligate Thermophiles
They can only grow at temperature above 37C. E.g. Archea, Eubacteria
Facultative Thermophiles
They can grow above and below 37C°.

61. 3. Oxygen
Obligate aerobes
Which always require oxygen to grow e.gPseudomonas
Obligate anaerobes
They don't require oxygen at all for growth and respiration e.gclostridium
Micro aerophiles
They grow best in small amount of oxygen e.gCampylobacter
Aerotolerent anaerobes
They can survive in presence of oxygen but don't use it in their metabolism.
e.gLactobacillus

62. Type of Organism Description Oxygen Level Example
Obligate aerobe With O2 20% B.subtilis
Obligate anaerobe Without O2 0% C. botulinum
Facultative anaerobe With/without
O2
0-20% E.coli
Microaerophiles With O2 2-10% Campylobacter
Aerotolerant With/without
O2
0-20%

63. 4.Moisture
Unlike large organisms that have protective covering and internal fluid environments
single celled are directly exposed to the environments so they need constant moisture
to survive.
5.Osmotic pressure
a) Plasmolysis happens when the dissolved substances in the environment exert more
pressure as compared to the substances within the cell (hyperosmotic environment)
and lead to cell shrink. b)Distension when cells in distilled water have higher osmotic
pressure inside as compared to the environment and gain water and cell become turgid
to prevent bursting. This is called distension.

64. 6.Radiation
Radiations like U V rays and gamma rays can mutate DNA and kill microorganisms,
the bacterium
Deinococcus radiodurans can survive harsh radiations such
microbes are used in cleaning up the contaminated sites.
7.Hydrostatic pressure
Many microbes have to face water pressure specially in lakes and oceans and
those who survive in such habitat are Barophiles.

65. Nutrition requirements
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Carbon sources:
Carbon is used as energy source.
Its compounds as building blocks to synthesize cell components
Nitrogen sources:
Microbes need nitrogen to synthesize enzymes, other proteins and nucleic acids.
Some obtain by inorganic sources.
They reduce nitrate ions to amino groups and use amino groups to make amino acids
then these are used in protein synthesis.

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Sulphur and phosphorus:
Microbes obtain sulfur from inorganic sulfate salts and sulphur containing amino
acids.
They use them to make proteins, coenzymes and other cell components.
Microbes obtain phosphorus mainly from inorganic phosphate ions.
They use phosphorus to synthesize ATP phospholipids and nucleic acids.

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Trace elements
Microbes require trace elements copper, iron, zinc, and cobalt usually in the form of
ions.
Trace elements serve as cofactors in enzymatic reactions potassium,
zinc magnesium and manganese are used to activate certain enzymes.
cobalt is required to synthesize vitamin B12
Iron is required for the synthesis of heme containing
compounds .
Calcium is required by gram positive bacteria for the synthesis of cell wall and by
spore forming organisms for the synthesis of spores.
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Vitamins
An organic compound that an organism require in small amount and used as a
coenzyme.
These include folic acid, vitamin B12, and vitamin k human pathogens require the
vitamins and then they become able to grow in the host by receiving these from host.
Microbes living in human intestine manufacture vitamin k which is necessary for blood
coagulation

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Environmental perspective
Environment is rich reservoir for the growth of microbes specially soil in which all
essential elements that are required for the growth of microbes are present.
Environment contains all types of habitats to support different categories of
microbes starting from pH, oxygen, temperature and pressures.
This mechanism is maintained by the biological processes of which the microbes are
a part, for example nitrogen and carbon cycling.
By isolating the microbes from environment and knowing the composition of their
habitat, now they are cultured in the laboratories by providing with all
nutrition requirement.